In vivo regeneration of oligodendrocytes by bolus injection of NGFβ

ABSTRACT

A method is presented for regenerating oligodendrocytes in diseases such as multiple sclerosis by administering human β nerve growth factor (NGF-β) by bolus injection. Treatment comprises 1-10 bolus injections in a dose of 0.05 to 5.0 μg/kg body at an interval of 1 to 21 days.

The present invention concerns a novel method for the regeneration ofoligodendrocytes, in particular, of human oligodendrocytes, and for thetreatment of diseases in which a demyelination of nerve fibers occurs, amethod of manufacturing a therapeutic agent, as well as the use of humanβ nerve growth factor (NGF) for said therapeutic use.

The covering of nerve fibers in the central nervous system (CNS) withmyelin is essential for the function of neuronal signal transmission.The myelin sheath is formed by oligodendrocytes (OL), the fibers ofwhich wrap around the axon of a nerve cell. Demyelinating diseases suchas multiple sclerosis in which the myelin sheath of the axon is damagedor destroyed also lead to impairments of the OL. However, the OL remainscapable of regenerating the myelin sheaths. Therefore, theidentification and characterization of factors which are responsible forincreased regeneration of OL is very important for the molecularunderstanding of demyelination diseases, such as multiple sclerosis(MS), and for the development of therapeutic agents.

It is known from PCT/EP92/01173 (17) that the regeneration ofoligodendrocytes is improved when they are treated with NGF or activefragments of NGF.

NGF is a neurotrophic factor which is well characterized. Its gene isdescribed in Ullrich et al. (1993) (1) and EP-B 0 121 338 (U.S. Pat. No.5,169,762) (13). The recombinant production of NGF from E.coli isdescribed in EP-A 0 544 293 (15), EP-A 0 450 386 (U.S. Pat. No.5,235,043) (14), EP-A 0 370 171 (16) and U.S. Ser. No. 08/266610 (18).

NGF has potent and beneficial effects on cholinergic neurons afteraxotomy (see, for example, Hoffman et al. (1990) (2)). NGF infusionsstimulate the regeneration of transsected cholinergic neurons (Gage(1988) (3), Tuszynski et al. (1990) (4)). It is further known thatinfusions of NGF stimulate the expression of choline acetyltransferase(ChAT) (Hefti et al. (1984) (5) and p75NGF receptor mRNA (Gage et al.(1989) (6)).

For the therapeutic application, NGF is infused for a period of severalweeks. Olson et al. (1991) (11) describes an infusion of NGF through anintraventricular cannula for 23 days with a total dose of 3.3 mg(corresponding to approximately 140 μg/24 hr) for the treatment ofParkinson's patients after having received fetal dopaminergic grafts. Itis also known to deliver NGF therapeutically in Alzheimer's disease viaa programmable pump planted subcutaneously into the abdominal wall andconnected by a subcutaneous catheter to the intraventricular catheter.To the patient a total of 6.6 mg of NGF was delivered during threemonths at a rate of 15 μl/hr (corresponding to approximately 75 μg/24 hr(Olson et al. (1992) (12))).

SUMMARY OF THE INVENTION

It was surprisingly found that for the regeneration of oligodendrocytes,i.e. in MS, an interval application leads to an improved and acceleratedremyelination of damaged nerve fibers as compared to continuousapplication of the therapeutic agent.

The invention concerns a method of preparing a therapeutic agent for thetreatment of multiple sclerosis, the method being characterized in thathuman β nerve growth factor (NGF) is brought into a pharmaceuticallyacceptable formulation for administering in a dose between 0.05 μg and 5μg/kg body weight in 1 to 10 bolus injections at an interval of 1 to 21days, preferably 1 to 12 days.

According to the invention, NGF is administered in a dose between 0.05μg and 5 μg/kg, 3.5 to 350 μg, preferably 3.5 to 210 μg per injection,at an interval of 1 to 21 days. Preferably, the dose is applied in 1 to10 injections.

In a preferred embodiment of the invention, it is preferred to use 0.01μg to 3 μg/kg, and more preferably 0.1 μg to 1 μg/kg.

It is preferred to administer NGF intrathecally, into the cerebrospinalfluid space of the ventricle or spinal cord, preferably of the lateralventricle or the lumbar spinal cord.

DETAILED DESCRIPTION OF THE INVENTION

The term “NGF” means β-unit of human NGF. β-NGF has an amino acidsequence of 118 amino acids and is present as a dimer in solution. Theamino acid and DNA sequence is described in Ullrich et al. (1993) (1).

The pharmaceutical compositions which are used according to theinvention and contain β-NGF may be administered in any sterilebiocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline or dextrose solution, preferably in an acidicsolution having a pH of about 4 to 5, preferably in an acetate buffer.The amount of NGF protein which will be effective in the treatment of MSis in a dose between 0.05 μg and 5 μg/kg body weight at an interval of 1to 21 days, preferably in 1 to 10 injections. It is further preferred touse 3.5 to 350 μg/injection.

Methods of introduction include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, oral,intrapeulmonal and intranasal administration. In addition, it may bedesirable to introduce the pharmaceutical composition of the inventioninto the central nervous system by any suitable route, includingintrathecal, e.g. intraventricular injection. Intraventricular injectionmay be facilitated by an intraventricular catheter, for example attachedto a reservoir, such as an implantable port catheter system, such as thePeriplant® filtrosafe (B. Braun GmbH, Spangenberg, DE).

Furthermore, it may be desirable to administer the pharmaceuticalcompositions, which are used according to the invention, locally to thearea in need of treatment. This may be achieved by, for example, and notby way of limitation, injection by means of a catheter or by means of animplant, said implant being of a porous, non-porous or gelatinousmaterial, including membranes, such as sialastic membranes or fibers.NGF is preferably administered during the relapse period. If high dosesare used, only a few or even one bolus application is sufficient.

The following examples and FIG. 1 are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

FIG. 1 shows coronal magnetic resonance tomography imaging (MRI) of pigbrains 7 days after lysolecithin-induced demyelination. a) Lesionedanimal, receiving sham-treatment with a single intraventricularinjection of 70 μg Cytochrom C. Arrows point to hyperintensity in thesubcortical white matter of the lesioned side (R—right hemisphere) ascompared to the non-lesioned left hemisphere (L). b) Identicallylesioned animal as shown in a), receiving a single intraventricularinjection of 70 μg NGF. Note that both hemispheres are symmetricallyconfigurated with no remaining hyperintensity in the lesioned side (R).

EXAMPLE 1 Experimental Design of an Animal Model of Demyelination

Lesion

An experimental model of demyelination was established in the female,adult “Göttingen Mini Pig” (age: 10-14 months, weight: 25-30 kg) bystereotaxic injection of lysolecithin (LL) at multiple sites into theperiventricular subcortical white matter of the brain (usually 2-3injection sites located in one hemisphere; 5 to 25 μl of 1% LL in 0.9%saline per lesion infused over a period of 15 min). This infusion causesa rapid reduction of myelin sheaths within the diffusion area of thedetergent (Blakemore (1978) (7)).

NGF-application

Immediately after LL application, a catheter system (PeriplantFiltrosafe®, Braun, Melsungen, Germany) is implanted either into thelateral ventricle of the brain or in the subarachnoidal space of thelumbar spinal cord (Motsch and Robert (1987) (8); Krames and Lanning(1993) (9)). Two ways of NGF application were used:

1) After fixation of the catheter with Ionocem® bone cement (Ionos,Seefeld, Germany) the intrathecal catheter is connected with asubcutaneous drug delivery system (Periplant Filtrosafe®, Braun,Melsungen, Germany) which is implanted subcutaneously in the upper backregion. NGF (dissolved in phosphate-buffered saline, pH 7.2) injectedevery 2nd day through the skin into the port, starting 1 day afterinitial LL lesions, by single bolus injections into the port at a doseof 0.05-3 μg/kg body weight (1.25 to 90 μg per injection, corresponds to3.5 to 210 μg per injection (human weight: 70 kg). 1-5 injections werecarried out into the cerebrospinal fluid within a period of 1-12 daysafter LL-induced demyelination.

2) The effect of single bolus injections of NGF described above wascompared with the continuous infusion of NGF into the lateral ventriclevia osmotic minipumps (Alzet® for 1-12 days at a dose of 0.5 μg/h).

Controls

Controls were carried out by replacement of NGF solution with an equaldose of Cytochrome C applied in the same ways and time interval asdescribed in 1) and 2).

Evaluation of NGF effects

At the end of the NGF (control, respectively) application period, theexperimental animals were sacrificed, the brain was removed and preparedfor histological examination: The areas of interest were studied withroutine histological (H.E. staining, Luxol Fast Blue staining),immunocytochemical (myelin basic protein—MBP) and electronmicroscopic(oligodendrocytes, myelin lamellae) procedures.

Findings

Injection of LL causes a widespread demyelination throughout the area ofdiffusion.

Chronic infusion of infusion of NGF via Alzet pumps: An improved andaccelerated remyelination of the lesioned myelin sheaths of nerve fiberswas detected within the application interval as compared to controls.Significant regeneration was observed 7 days after initial lesionwhereas severe demyelination was still present in the white matter ofsham-treated animals.

Bolus NGF injection every 2nd day: Significant regeneration was alsonoted under NGF treatment in this system, with no apparent differencesto animals receiving chronic infusions of NGF. Furthermore, NGF injectedin the CSF space in the area of the spinal cord showed the same positiveeffect on remyelination as found for intraventricular injections.

The data demonstrate that injections of NGF into the cerebrospinal fluidlead to an improved and accelerated remyelination, most likely due toregeneration and proliferation of oligodendrocytes. Compared to chronicNGF infusion with the help of pumps, the application via anintrathecally implanted port allows an easy-to-handle, save andinexpensive way of supplying the brain with the neurotrophin at anadjustable and optimal concentration for maximal therapeutic efficacy.In addition, in patients with multiple sclerosis suffering from repeatedattacks (i.e. relapsing-remitting MS) immediate interval treatments atthe time of the attack within a period of several years are possible,minimizing the risk of complications (i.e. infections) due to necessarychanges of catheter systems or refills of pumps needed during chronic orrepeated infusions.

Additional Supporting Data

There is a clear correlation between the degree of remyelination andfunctional improvements in the state of demyelinating diseases (IFNβstudy group (1993) (10)).

Data obtained from animal models using pigs have a great clinicalrelevance in biomedical research, i.e. numerous studies on thecardiovascular system, skin and peripheral nervous system that haveproven that data from porcine tissue are applicable on human subjects.

List of References

(1) Ullrich et al., Nature 303 (1993) 821

(2) Hoffman et al., Exp. Neurol. 110 (1990) 39-44

(3) Gage, F. H., J. Comp. Neurol. 269 (1988) 147-155

(4) Tuszynski, M. H., et al., Neuroscience 36 (1990) 33-44

(5) Hefti, F., et al., Brain Res. 293 (1984) 305-311

(6) Gage, F. H., et al., Neuron 2 (1989) 1177-1184

(7) Blakemore, W. F., Neuropathol. Appl. Neurobiol. 4 (1978) 47-59

(8) Motsch, J., and Robert, B., Schmerz 3 (1987) 115-125

(9) Krames, E. S., Lanning, R. M., J. Pain Symptom Manage. 8 (1993)539-548

(10) The IFNβ multiple sclerosis study group, Neurology 43 (1993)662-667

(11) Olson, L., et al., Arch. Neurol. 48 (1991) 373-381

(12) Olson, L., et al., J. Neural. Transm. [P-D Sect] 4 (1992) 79-95

(13) EP-B 0 121 338 (U.S. Pat. No. 5,169,762)

(14) EP-A 0 450 386 (U.S. Pat. No. 5,235,043)

(15) EP-A 0 544 293

(16) EP-A 0 370 171

(17) PCT/EP92/01173

(18) U.S. Ser. No. 08/266,610

What is claimed is:
 1. A method of regenerating oligodendrocytes for thetreatment of a disease in a human in which a demyelination of nervefibers occurs, comprising the steps of bringing human β nerve growthfactor into a pharmaceutically acceptable formulation for administrationto a human; and administering the human β nerve growth factor by bolusinjection to the human, wherein each bolus injection is in an amount ofthe human β nerve growth factor of 0.05 μg to 5 μg/kg body weight of thehuman, and wherein the administration involves 1 to 10 bolus injectionsover a time interval of 1 to 21 days.
 2. The method of claim 6, whereineach bolus injection has an amount of the human β nerve growth factor of0.1 μg to 3 μg/kg body weight of the human.
 3. The method of claim 1,wherein each bolus injection has an amount of the human β nerve growthfactor of 0.1 μg to 1 μg/kg body weight of the human.
 4. The method ofclaim 1, wherein the human β nerve growth factor is administered in adose from 3.5 μg to 350 μg/bolus injection.
 5. The method of claim 1,wherein the human β nerve growth factor is administered in a dose from3.5 μg to 210 μg/bolus injection.
 6. The method of claim 1, wherein thehuman β nerve growth factor is administered over a time interval of 1 to12 days.
 7. The method of claim 1, wherein the human β nerve growthfactor is administered intrathecally into the cerebrospinal fluid spaceof the ventricle or spinal cord of the human.
 8. The method of claim 7,wherein the human β nerve growth factor is administered into the lateralventrical or the lumbar spinal cord.
 9. The method according to claim 7,wherein the administration of the human β nerve growth factor into thecerebrospinal fluid is an intraventricular bolus injection carried outvia an intraventricular catheter attached to an implanted port forinjection.
 10. The method of claim 6, wherein the human β nerve growthfactor is administered intradermally, intramuscularly,intraperitoneally, intravenously, or subcutaneously.
 11. The method ofclaim 6, wherein the human β nerve growth factor is administered bycatheter to an area of the human in need of treatment.